Ubiquitin conjugating enzymes 8 and 9

ABSTRACT

Human UCE 7, UCE 8 and UCE 9 polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods of utilizing such polypeptide for the treatment of the proliferation of malignant cells. Antagonists against such polypeptides and their uses as a therapeutic to treat Alzheimer&#39;s disease, atrophying skeletal muscle, African swine fever virus and apoptotic cell death are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in UCE 7, 8 and 9 nucleic acid sequences and the concentration of polypeptides encoded by such sequences.

This application is entitled to the benefits of 35 USC §120 for prioritybased on PCT/US95/01250, filed 31 Jan. 1995.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptides of the presentinvention are Ubiquitin Conjugating Enzymes 7, 8 and 9, sometimeshereinafter referred to as "UCE 7, 8 and 9." The invention also relatesto modulating the action of such polypeptides.

Mammalian cells contain two distinct proteolytic pathways that areinvolved in different aspects of protein breakdown. One of these isubiquitin-dependent, it is a major pathway in eukaryotes involved in theselective degradation of abnormal and short-lived proteins. Ubiquitin isa highly conserved 76 amino acid residue protein present in eukaryoticcells either free or covalently attached to a great variety of proteins.The post-translational attachment of ubiquitin to other proteins iscatalyzed by ubiquitin conjugating enzymes and involves the formation ofan isopeptide bond between the C-terminal glycine residue of ubiquitinand the epsilon-amino group of a lysine-residue in an acceptor protein.

Ubiquitin-protein conjugation is highly selective and is required for asurprising variety of cellular functions. Genetic studies in yeastshowed that ubiquitin conjugating enzymes are required for DNA repair,induced mutagenesis, sporulation, repression of retrotransposition, cellcycle progression, cell viability, heat shock resistance, cadmiumtolerance, and peroxisome biogenesis. Several in vivo substrates havebeen identified, including histones, actin, cell surface receptors, theMATα2 transcriptional repressor, the tumor suppressor protein p53, theMos kinase and cyclins. UCE 7, 8 and 9 may play a major role inselective protein degradation in human cells.

The ubiquitin gene is one of the genes known to be stimulated during theapoptotic death program and ubiquitin of nuclear proteins might beinvolved in chromatin disorganization and oligonucleosomalfragmentation, which are among the key events occurring in apoptosis.Apoptosis, the classical type of programmed cell death, can be triggeredin many cell types by widely diverse stimuli, for example, gamma rays atlow doses can induce apoptosis in vitro in interphase human lymphocytes.In this type of apoptosis induction, activated gene expression isnecessary for the fulfillment of the death program. It has been reported(Delic, J., et al., Mol. Cell Biol., 13:4875, 83 (1993)) that there is arelationship between ubiquitin gene expression or ubiquitination andgamma-irradiation-mediated apoptosis in normal circulating humanlymphocytes. In this report it has been demonstrated that the ubiquitinmRNA level is increased as a consequence of the activation of ubiquitingene transcription 15 to 90 minutes after initiation of apoptosis;specifically, in apoptotic cells, and not in all irradiated cells,nuclear proteins are highly ubiquitinated; and ubiquitinsequence-specific antisense oligonucleotide inhibition results in adecreased level of ubiquitinated nuclear proteins and considerablydiminishes the proportion of cells exhibiting the apoptotic deathpattern.

Perturbations of ubiquitin system can also induce a programmed necroticresponse in plants such as leaf curling, vascular tissue alterations andnecrotic lesions.

Ubiquitin can inhibit the cytotoxic properties of platelets and theproduction of oxygen metabolites by these cells. Moreover, this moleculeis able to act as a proaggregating factor and seems of a great interestin pathologies involving defects in platelet aggregation. Ubiquitin alsoplays a role in the regulation of immunological disorders in whichplatelets seem to be implicated such as hymenoptera venomhypersensitivity and aspirin-sensitive asthma, since in both situations,ubiquitin is able to inhibit the cytotoxic function of platelets.

Ubiquitin has also been shown to be increased in patients withAlzheimer's disease (Taddei, N., et al., Neurosci. Lett., 151:158-61(1993)). This study concerned the amount of soluble ubiquitin indifferent cortical and subcortical regions of brains from patients withAlzheimer's disease compared to the amount in a normal brain. Thesoluble ubiquitin content was significantly higher in pathologicaltissue than in normal tissue. The primary structure of ubiquitinisolated from brain tissue affected by Alzheimer's degenerativeprocesses was determined and resulted to be identical to normal humanubiquitin. This report suggests that an impairment of the process ofintracellular, ubiquitin-dependent proteolysis might play an importantrole in the pathogenesis of this neurodegenerative disease.

Ubiquitin-proteasome system also plays a major role in specificprocessing and subsequent presentation of MHC class I-restrictedantigens.

Maturation of the p105 NF-kB precursor into the active p50 subunit ofthe transcriptional activator also proceeds in a ubiquitin andproteasome-dependent manner. Furthermore, inhibitors to the proteasomeblock degradation of IkBa and thus prevent tumor necrosis factor alphainduced activation of NF-kB and its entry into the nucleus.

The unstable c-Jun, but not the stable v-Jun, is multi-ubiquitinated anddegraded. The escape of the oncogenic v-Jun from ubiquitin-dependentdegradation suggests a route to the malignant transformation. Anotherproto-oncoprotein, c-Mos, is also degraded by the ubiquitin system.

The human papilloma virus (HPV) derived E6 proteins stimulate ATP andubiquitin dependent conjugation and degradation of p53, such a mechanismcould explain the extremely low levels of p53 observed inHPV-transformed cervical carcinoma lines and propose a mechanism for thetumorigenicity of these onco-proteins.

Several cell surface receptors, including the lymphocyte homingreceptor, growth hormone receptor, and growth factor receptor (PDGF,steel factor) were also found to be modified by ubiquitin.

The polypeptides of the present invention have been putativelyidentified as UCE 7, 8 and 9. This identification has been made as aresult of amino acid sequence homology.

In accordance with one aspect of the present invention, there areprovided novel mature polypeptides which are UCE 7, 8 and 9, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof. The polypeptides of thepresent invention are of human origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding UCE 7, 8 and 9,including mRNAs, DNA's, cDNA's, genomic DNA, as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptides by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a human UCE 7, 8 or 9 nucleic acidsequence, under conditions promoting expression of said proteins andsubsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, to treat malignant transformations, immunological disorders, tomark unwanted cells for cell death, and to screen for agonists andantagonists which interact with the polypeptides.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be employed toinhibit the action of such polypeptides, for example, in the treatmentof atrophying skeletal muscle, cervical carcinoma and certain tumors,Alzheimer's disease, endemic pemphigus foliaceus and African swinefever.

In accordance with another aspect of the present invention, there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to UCE 7, 8 and 9 sequences.

In accordance with another aspect of the present invention, there isprovided a process for utilizing such polypeptides, or polynucleotidesencoding such polypeptides, for in vitro purposes related to scientificresearch, synthesis of DNA and manufacture of DNA vectors.

In accordance with yet another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to mutations in UCE 7, 8 or 9 nucleic acid sequencesor over-expression of the polypeptides encoded by such sequences.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 illustrates the cDNA sequence and the corresponding deduced aminoacid sequence of UCE 7 polypeptide. The standard one-letterabbreviations for amino acids is used. Sequencing was performed using a373 Automated DNA sequencer (Applied Biosystems, Inc.). Sequencingaccuracy is predicted to be greater than 97% accurate.

FIG. 2 illustrates the cDNA sequence and the corresponding deduced aminoacid sequence of UCE 8 polypeptide.

FIG. 3 illustrates the cDNA sequence and the corresponding deduced aminoacid sequence of UCE 9 polypeptide.

FIG. 4 illustrates the amino acid sequence homology between UCE 7 andUCE from Drosophila melanogaster.

FIG. 5 illustrates the amino acid sequence homology between UCE 8 andthe Caenorhabditis elegans UCE gene product.

FIG. 6 illustrates the amino acid sequence homology between UCE 9 andUCE from Saccharomyces cerevisiae.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptides having the deduced amino acid sequence of FIGS. 1, 2 and 3(SEQ ID No. 2, 4 and 6), or for the mature polypeptides encoded by thecDNAs of the clone deposited as ATCC Deposit No. 75877, 75876 and 75878on Aug. 29, 1994 encoding UCE 7, 8 and 9, respectively.

The ATCC numbers referred to above are directed to biological depositswith the ATCC, 12301 Parklawn Drive, Rockville, Md. 20852. Since thestrains referred to are being maintained under the terms of the BudapestTreaty, each of them will be made available to a patent office signatoryto the Budapest Treaty.

A polynucleotide encoding a UCE 7 polypeptide of the present inventionmay be obtained from tumor testis, activated T-cells and chondrosarcoma.The polynucleotide of this invention was discovered in a cDNA libraryderived from Raji cells (cycloheximide treated). It is structurallyrelated to the human ubiquitin conjugating enzyme family. It contains anopen reading frame encoding a protein of 147 amino acid residues. Theprotein exhibits the highest degree of homology to UCE from Drosophilamelanogastor with 93% identity and 96% similarity over a 147 amino acidstretch.

A polynucleotide encoding a UCE 8 polypeptide of the present inventionmay be obtained from osteoclastoma, tumor testis and activated T-cells.The polynucleotide of this invention was discovered in a cDNA libraryderived from human fetal brain. It is structurally related to the humanubiquitin conjugating enzyme family. It contains an open reading frameencoding a protein of 154 amino acid residues. The protein exhibits thehighest degree of homology to UCE from Caenorhabditis elegans with 55%identity and 78% similarity over a 154 amino acid stretch.

A polynucleotide encoding a UCE 9 polypeptide of the present inventionmay be obtained from embryo, smooth muscle and greater omentum. Thepolynucleotide of this invention was discovered in a cDNA libraryderived from human greater omentum. It is structurally related to thehuman ubiquitin conjugating enzyme family. It contains an open readingframe encoding a protein of 193 amino acid residues. The proteinexhibits the highest degree of homology to UCE from S. cerevisiae with61% identity and 72% similarity over a 193 amino acid stretch.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIGS. 1, 2 and 3 (SEQ ID No.1, 3 and 5) or that of the deposited clone or may be a different codingsequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same mature polypeptides asthe DNA of FIGS. 1, 2 and 3 (SEQ ID No. 1, 3 and 5) or the depositedcDNA.

The polynucleotide which encodes for the mature polypeptides of FIGS. 1,2 and 3 (SEQ ID No. 2, 4 and 6) or for the mature polypeptide encoded bythe deposited cDNA may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5' and/or 3' of the coding sequence forthe mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptidesas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequencesof FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the polypeptides encodedby the cDNA(s) of the deposited clone. The variant of the polynucleotidemay be a naturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptides as shown in FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6)or the same mature polypeptide encoded by the cDNA(s) of the depositedclone as well as variants of such polynucleotides which variants encodefor a fragment, derivative or analog of the polypeptides of FIGS. 1, 2and 3 (SEQ ID No. 2, 4 and 6) or the polypeptides encoded by the cDNA(s)of the deposited clone(s). Such nucleotide variants include deletionvariants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequencesshown in FIGS. 1, 2 and 3 (SEQ ID No. 1, 3 and 5) or of the codingsequence of the deposited clones. As known in the art, an allelicvariant is an alternate form of a polynucleotide sequence which may havea substitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptides.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptides of the present invention. The markersequence may be a hexahistidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptides fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term"stringent conditions" means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to UCE 7, 8 and 9 polypeptideswhich have the deduced amino acid sequence of FIGS. 1, 2 and 3 (SEQ IDNo. 2, 4 and 6) or which has the amino acid sequence encoded by thedeposited cDNAs, as well as fragments, analogs and derivatives of suchpolypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptides of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encodedby the deposited cDNA(s), means polypeptides which retain essentiallythe same biological function or activity as such polypeptides. Thus, ananalog includes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

The fragment, derivative or analog of the polypeptides of FIGS. 1, 2 and3 (SEQ ID No. 2, 4 and 6) or that encoded by the deposited cDNA(s) maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol). Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular, the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the UCE 7, 8 and 9 genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotides may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the polypeptide.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2. andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation, initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The UCE 7, 8 and 9 polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The UCE 7, 8 and 9 polypeptides and agonists and antagonists which arepolypeptides, described below, may also be employed in accordance withthe present invention by expression of such polypeptides in vivo, whichis often referred to as "gene therapy".

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, arian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Once the UCE 7, 8 and 9 polypeptides are being expressedintra-cellularly via gene therapy, they may be employed to provide asignal for the lymphocyte homing receptor thereby regulating lymphocytetrafficking. The growth hormone receptor also utilizes ubiquitin tosignal ligands, and, therefore, the UCE 7, 8 and 9 polypeptides may beemployed to regulate activation of the growth receptor.

UCE 7, 8 and 9 polypeptides may be employed to overcome many viralinfections by overcoming the suppressed programmed cell death induced bythese viruses, since programmed cell death may be one of the primaryantiviral defense mechanisms of cells.

UCE 7, 8 and 9 polypeptides may also be employed to treatimmuno-suppression related disorders, such as AIDS, by targeting virusinfected cells for cell death.

UCE 7, 8 and 9 may also be employed to inhibit the cytotoxic propertiesof platelets and the production of oxygen metabolites by platelets.These polypeptides may also be employed to regulate immunologicaldisorders in which platelets seem to be involved, for example,hymenoptera venom hypersensitivity and aspirin-sensitive asthma.

UCE 7, 8 and 9 may also be employed to treat malignant transformationbecause proto-oncoproteins c-Mos and v-Jun are degraded in aubiquitin-dependent manner.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA, manufacture of DNAvectors and for the purpose of providing diagnostics and therapeuticsfor the treatment of human disease.

The present invention further provides a method of screening compoundsto identify those which enhance (agonists) or block (antagonists) theactivity of the UCE 7, 8 and 9 enzymes. An example of such a methodcomprises combining reactants in the presence of UCE 7, 8 or 9 and acompound to be screened under conditions where ubiquitin is normallytransferred to a protein substrate. The reactants comprise [I¹²⁵]ubiquitin, ATP, and a protein substrate, for example, histones. Undernormal conditions, ubiquitin would be transferred to the proteinsubstrate and this transfer is catalyzed by the UCE 7, 8 or 9 enzymes.The amount of labeled substrate, i.e., substrate with labeled ubiquitinattached thereto, could then be measured to determine if the compound tobe screened enhanced or blocked the catalysis of this reaction by UCE 7,8 or 9.

Human UCE 7, 8 and 9 are produced and function intra-cellulary,therefore, any antagonists must be intra-cellular. Examples of potentialUCE 7, 8 or 9 antagonists include antibodies which are producedintra-cellularly. For example, an antibody identified as antagonizingUCE 7, 8 and 9 may be produced intra-cellularly as a single chainantibody by procedures known in the art, such as transforming theappropriate cells with DNA encoding the sigle chain antibody to preventthe function of UCE 7, 8 or 9.

Another potential antagonist is an antisense construct prepared usingantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5' coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix--see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of UCE 7, 8 and 9. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the UCE 7, 8 and 9 polypeptide(antisense--Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of UCE 7, 8 and 9.

Yet another potential antagonist includes a mutated form, or mutein, ofUCE 7, 8 or 9 which recognizes substrate but does not catalyzeubiquitination and, therefore, acts to prevent UCE 7, 8 or 9 fromfunctioning.

Potential antagonists also include small molecules which are able topass through cell membranes and bind to the catalytic site of thepolypeptide thereby making the catalytic site inaccessible to substratesuch that normal biological activity is prevented.

The antagonists may be employed to treat a host of diseases in which UCE7, 8 and 9 catalyze the transfer of ubiquitin to a substrate and marksthat substrate for cell death. An example is Alzheimer's disease,wherein the ubiquitin content is found to be significantly higher intissue derived from patients with Alzheimer's disease than in normaltissue.

The antagonists may also be employed to treat atrophying skeletal musclesince the atrophying of this muscle occurs due to these cells beingmarked for cell death with ubiquitin.

The antagonists may also be employed to treat a patient infected withthe African swine fever virus which proliferates in the host andproduces a conjugating enzyme to kill cells in a attempt to overtake thehost's regulatory mechanisms. Accordingly, inhibiting UCE 7, 8 and 9prevents the proliferation of the African swine fever virus.

The antagonists may also be employed to treat a blistering skin diseasecalled endemic pemphigus foliaceus (EPF) which is also a type ofautoimmune disorder, which is thought to degrade skin through the use ofa ubiquitin conjugating enzyme.

The antagonists may also be employed to treat certain malignanttransformations, for example, Human papilloma virus (HPV) transformedcervical carcinoma which stimulates the ubiquitin-dependent degradationof p53, a tumor suppressor protein. Further, epidermal tumors in micehave been found to over-express ubiquitin genes.

The small molecule agonists and antagonists may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the agonists orantagonists and a pharmaceutically acceptable carrier or excipient. Sucha carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10 μg/kgbody weight and in most cases they will be administered in an amount notin excess of about 8 mg/Kg body weight per day. In most cases, thedosage is from about 10 μg/kg to about 1 mg/kg body weight daily, takinginto account the routes of administration, symptoms, etc.

This invention is also related to the use of the UCE genes as adiagnostic. Detection of a mutated form of UCE 7, 8 or 9 will allow adiagnosis of a disease or a susceptibility to a disease which resultsfrom underexpression of these genes, for example, tumors and immunedisorders.

Individuals carrying mutations in the human UCE 7, 8 or 9 gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding UCE 7, 8 or 9 can beused to identify and analyze mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled UCE 7, 8 or 9 RNA oralternatively, radiolabeled UCE 7, 8 or 9 antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of UCE 7, 8 or 9 protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples can detect the presence of tumors. Assays used to detect levelsof UCE 7, 8 or 9 protein in a sample derived from a host are well-knownto those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis and preferably anELISA assay. An Elisa assay initially comprises preparing an antibodyspecific to the UCE 7, 8 or 9 antigen, preferably a monoclonal antibody.In addition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or in this example a horseradishperoxidase enzyme. A sample is now removed from a host and incubated ona solid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like BSA. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attach to any UCE 7, 8 or 9 proteins attached to thepolystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to UCE 7, 8 or 9 protein. Unattached reporterantibody is then washed out. Peroxidase substrates are then added to thedisband the amount of color developed in a given time period is ameasurement of the amount of protein present in a given volume ofpatient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to UCE7, 8 or 9 protein are attached to a solid support and labeled UCE 7, 8or 9 and a sample derived from the host are passed over the solidsupport and the amount of label detected attached to the solid supportcan be correlated to a quantity in the sample.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment, asdescribed in Sambrook et al., Molecular Cloning: A laboratory Manual,Second Edition, Cold Spring Harbor, N.Y. (1989).

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of UCE 7

The DNA sequence encoding UCE 7, ATCC # 75877, was initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' sequences ofthe processed UCE 7 protein and the vector sequences 3' to the UCE 7gene. Additional nucleotides corresponding to UCE 7 were added to the 5'and 3' end sequences respectively. The forward oligonucleotide primerhas the sequence 5' CCCGGATCCGCTTCGAAGAGAATCCACAAG 3' (SEQ ID No. 7)contains a Bam HI restriction enzyme site followed by 21 nucleotides ofUCE 7 coding sequence starting from the presumed terminal amino acid ofthe processed protein codon. The reverse primer 5'GCGCAAGCTTTTACATCGCATACTTCTGAGTCC 3' (SEQ ID No. 8) contains a Hind IIIsite, a stop codon plus 20 nucleotides of UCE 7. The restriction enzymesites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and single restriction enzyme sites. pQE-9 wasthen digested with Bam H1 and Hind III. The amplified sequences wereligated into pQE-9 and were inserted in frame with the sequence encodingfor the histidine tag and the RBS. The ligation mixture was then used totransform E. coli strain m15/pREP4 (Qiagen) by the procedure describedin Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Laboratory Press, (1989). M15/pREP4 contains multiple copies ofthe plasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis.

Clones containing the desired constructs were grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells were grown to an optical density600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized UCE 7 was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). UCE 7 was eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 molar glutathione (oxidized). After incubation in thissolution for 12 hours the protein was dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 2 Bacterial Expression and Purification of UCE 8

The DNA sequence encoding UCE 8, ATCC # 75876, was initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' sequences ofthe processed UCE 8 protein and the vector sequences 3' to the UCE 8gene. Additional nucleotides corresponding to UCE 8 were added to the 5'and 3' end sequences, respectively. The forward oligonucleotide primerhas the sequence 5' CCCGGATCCGCGGCCAGCAGGAGGCTGATG 3' (SEQ ID No. 9)contains a Bam HI restriction enzyme site followed by 21 nucleotides ofUCE 8 coding sequence starting from the presumed terminal amino acid ofthe processed protein codon. The reverse primer 5'GCGCAAGCTTTTAGTCCACAGGTCG 3' (SEQ ID No. 10) contains a Hind III site, astop codon plus 15 nucleotides of UCE 8 coding sequence. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and single restriction enzyme sites. pQE-9 wasthen digested with Bam H1 and Hind III. The amplified sequences wereligated into pQE-9 and were inserted in frame with the sequence encodingfor the histidine tag and the RBS. The ligation mixture was then used totransform E. coli strain m15/pREP4 (Qiagen) by the procedure describedin Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Laboratory Press, (1989). M15/pREP4 contains multiple copies ofthe plasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis.

Clones containing the desired constructs were grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells were grown to an optical density600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized UCE 8 was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). UCE 8 was eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein was dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 3 Bacterial Expression and Purification of UCE 9

The DNA sequence encoding UCE 9, ATCC # 75878, is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' sequences ofthe processed UCE 9 protein and the vector sequences 3' to the UCE 9gene. Additional nucleotides corresponding to UCE 9 are added to the 5'and 3' end sequences respectively. The forward oligonucleotide primerhas the sequence 5' GCGCGGATCCACAGTCCAAGCACTAGGGC 3' (SEQ ID No. 11)contains a Bam HI restriction enzyme site followed by 19 nucleotides ofUCE 9 coding sequence starting from the presumed terminal amino acid ofthe processed protein codon. The reverse primer 5'GCGCAAGCTTCTATGTGGCGTACCGCTTGG 3' (SEQ ID No. 12) contains complementarysequences to a Hind III site and is followed by 20 nucleotides of UCE 9including the translational stop codon. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311).pQE-9 encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with Bam H1 and Hind III. The amplified sequencesare ligated into pQE-9 and are inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture is thenused to transform E. coli strain m15/pREP4 (Qiagen) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989). M15/pREP4 containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") is then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells are grown an extra 3 to 4 hours. Cellsare then harvested by centrifugation. The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized UCE 9 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). UCE 9 is eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 molar glutathione(reduced) and 2 molar glutathione (oxidized). After incubation in thissolution for 12 hours the protein is dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 4 Cloning and Expression of UCE 7 Using the BaculovirusExpression System

The DNA sequence encoding the full length UCE 7 protein, ATCC # 75877,is amplified using PCR oligonucleotide primers corresponding to the 5'and 3' sequences of the gene:

The forward primer has the sequence 5' GCGCGGATCCACCAT GGCTCTGAAGAGAATCC3' (SEQ ID No. 13) and contains a Bam HI restriction enzyme site (inbold) followed by 3 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) which is just behind the first 15 nucleotides of theUCE 7 gene (the initiation codon for translation "ATG" is underlined).

The reverse primer has the sequence 5' GCGCTCTAGATTACATCGCATACTTCTGAGTCC 3' (SEQ ID No. 14) and contains the cleavage site forthe restriction endonuclease XbaI and 23 nucleotides complementary tothe 3' translated sequence of the UCE 7 gene. The amplified sequencesare isolated from a 1% agarose gel using a commercially available kit("Geneclean," BIO 101 Inc., La Jolla, Calif.). The fragment is thendigested with the endonucleases Bam HI and XbaI and then purified againon a 1% agarose gel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the UCE 7 protein using the baculovirus expressionsystem (for review see: Summers, M. D. and Smith, G. E. 1987, A manualof methods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases Bam HI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pac373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes Bam HI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit ("Geneclean" BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E. coli DH5α cells are then transformed and bacteria identifiedthat contained the plasmid (pBac UCE7) with the UCE 7 gene using theenzymes Bam HI and XbaI. The sequence of the cloned fragment isconfirmed by DNA sequencing.

5 μg of the plasmid pBac UCE 7 is cotransfected with 1.0 μg of acommercially available linearized baculovirus ("BaculoGold™ baculovirusDNA", Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac UCE7 aremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seededin a 35 mm tissue culture plate with 1 ml Grace's medium without serum.The plate is rocked back and forth to mix the newly added solution. Theplate is then incubated for 5 hours at 27° C. After 5 hours thetransfection solution is removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum is added. The plateis put back into an incubator and cultivation continued at 27° C. forfour days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with "Blue Gal" (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a "plaque assay" can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculoviruses is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-UCE-7 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 5 Cloning and Expression of UCE 8 Using the BaculovirusExpression System

The DNA sequence encoding the full length UCE 8 protein, ATCC # 75876,is amplified using PCR oligonucleotide primers corresponding to the 5'and 3' sequences of the gene:

The forward primer has the sequence 5' GCGCGGATCCACCATGGCGGCCAGCAGGAGGCT 3' (SEQ ID No. 15) and contains a Bam HI restrictionenzyme site (in bold) followed by 3 nucleotides resembling an efficientsignal for the initiation of translation in eukaryotic cells (Kozak, M.,J. Mol. Biol., 196:947-950 (1987) and which is just behind the first 17nucleotides of the UCE 8 gene (the initiation codon for translation"ATG" is underlined).

The reverse primer has the sequence 5' GCGCTCTAGATTAGT CCACAGGTCG 3'(SEQ ID No. 16) and contains the cleavage site for the restrictionendonuclease XbaI and 15 nucleotides complementary to the 3' translatedsequence of the UCE 8 gene. The amplified sequences are isolated from a1% agarose gel using a commercially available kit ("Geneclean," BIO 101Inc., La Jolla, Calif.). The fragment is then digested with theendonucleases Bam HI and XbaI and then purified again on a 1% agarosegel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the UCE 8 protein using the baculovirus expressionsystem (for review see: Summers, M. D. and Smith, G. E. 1987, A manualof methods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases Bam HI, SmaI,XbaI, BglII and Asp718. The polyadenylation site of the simian virus(SV)40 is used for efficient polyadenylation. For an easy selection ofrecombinant viruses the beta-galactosidase gene from E. coli is insertedin the same orientation as the polyhedrin promoter followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for the cell-mediatedhomologous recombination of cotransfected wild-type viral DNA. Manyother baculovirus vectors could be used in place of pRG1 such as pac373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes Bam HI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit ("Geneclean" BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E. coli HB101 cells are then transformed and bacteria identifiedthat contained the plasmid (pBac UCE 8) with the UCE 8 gene using theenzymes Bam HI and XbaI. The sequence of the cloned fragment isconfirmed by DNA sequencing.

5 μg of the plasmid pBac UCE 8 is cotransfected with 1.0 μg of acommercially available linearized baculovirus ("BaculoGold™ baculovirusDNA", Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac UCE 8 aremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seededin a 35 mm tissue culture plate with 1 ml Grace' medium without serum.The plate is rocked back and forth to mix the newly added solution. Theplate is then incubated for 5 hours at 27° C. After 5 hours thetransfection solution is removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum is added. The plateis put back into an incubator and cultivation continued at 27° C. forfour days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with "Blue Gal" (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a "plaque assay" can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculoviruses is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-UCE 8 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 6 Cloning and Expression of UCE 9 Using the BaculovirusExpression System

The DNA sequence encoding the full length UCE 9 protein, ATCC # 75878,is amplified using PCR oligonucleotide primers corresponding to the 5'and 3' sequences of the gene:

The forward primer has the sequence 5' GATCGGATCCACCAT GACAGTCCAAGCACTAG3' (SEQ ID No. 17) and contains a Bam HI restriction enzyme site (inbold) followed by 3 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) and just behind the first 15 nucleotides of the UCE 9gene (the initiation codon for translation "ATG" is underlined).

The reverse primer has the sequence 5' GCGCTCTAGACTATG TGGCGTACCGCTTGG3' (SEQ ID No. 18) and contains the cleavage site for the restrictionendonuclease XbaI and 20 nucleotides complementary to the 3' translatedsequence of the UCE 9 gene. The amplified sequences are isolated from a1% agarose gel using a commercially available kit ("Geneclean," BIO 101Inc., La Jolla, Calif.). The fragment is then digested with theendonucleases Bam HI and XbaI and then purified again on a 1% agarosegel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the UCE 9 protein using the baculovirus expressionsystem (for review see: Summers, M. D. and Smith, G. E. 1987, A manualof methods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases Bam HI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pac373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes Bam HI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit ("Geneclean" BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E. coli DH5α cells are then transformed and bacteria identifiedthat contained the plasmid (pBac UCE9) with the UCE 9 gene using theenzymes Bam HI and XbaI. The sequence of the cloned fragment isconfirmed by DNA sequencing.

5 μg of the plasmid pBac UCE 9 is cotransfected with 1.0 μg of acommercially available linearized baculovirus ("BaculoGold™ baculovirusDNA", Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac UCE 9 aremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seededin a 35 mm tissue culture plate with 1 ml Grace's medium without serum.The plate is rocked back and forth to mix the newly added solution. Theplate is then incubated for 5 hours at 27° C. After 5 hours thetransfection solution is removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum is added. The plateis put back into an incubator and cultivation continued at 27° C. forfour days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with "Blue Gal" (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a "plaque assay" can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculoviruses is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-UCE 9 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 7 Expression of Recombinant UCE 7 in COS Cells

The expression of plasmid, UCE 7 HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire UCE 7 gene and an HA tag fused in frame toits 3' end is cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag correspond to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).The infusion of HA tag to the target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding UCE 7, ATCC # 75877, is constructed by PCRusing two primers: the 5' primer 5' GCGCGGATCCACCATGGCTCTGAAGAGAATCC 3'(SEQ ID No. 19) has a Bam HI site followed by 15 nucleotides of UCE 7coding sequence starting from the initiation codon. The reverse primer5' GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGACATCGCATACTTCTGAG3' (SEQ IDNo. 20) contains complementary sequences to an XbaI site, translationstop codon, HA tag and the last 17 nucleotides of the UCE 7 codingsequence (not including the stop codon). Therefore, the PCR productcontains a Bam HI site, UCE 7 coding sequence followed by HA tag fusedin frame, a translation termination stop codon next to the HA tag, andan XbaI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,are digested with Bam HI and XbaI restriction enzyme and ligated. Theligation mixture is transformed into E. coli strain SURE (StratageneCloning Systems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantUCE 7, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the UCE 7-HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵ S-cysteine two days post transfection. Culture mediais then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 8 Expression of Recombinant UCE 8 in COS Cells

The expression of plasmid, UCE 8-HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entir UCE 8 precursor and a HA tag fused in frameto its 3' end is cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag correspond to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).The infusion of HA tag to our target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding UCE 8, ATCC # 75876, is constructed by PCRusing two primers: the 5' primer 5' GCGCGGATCCACCATGGCGGCCAGCAGGAGGC 3'(SEQ ID No. 21) and contains a Bam HI site followed by 3 nucleotidesresembling a Kozak sequence plus 19 nucleotides of UCE 8 coding sequencestarting from the initiation codon; the 3' sequence 5'GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGTCCACAGGTCG 3' (SEQ ID No. 22)contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 12 nucleotides of the UCE 8 coding sequence(not including the stop codon). Therefore, the PCR product contains aBam HI site, UCE-8 coding sequence followed by HA tag fused in frame, atranslation termination stop codon next to the HA tag, and an XbaI site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith Bam HI and XbaI restriction enzyme and ligated. The ligationmixture is transformed into E. coli strain SURE (Stratagene CloningSystems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantUCE-8, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the UCE 8-HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵ S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 9 Expression of Recombinant UCE 9 in COS Cells

The expression of plasmid, pUCE 9-HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire UCE 9 protein and a HA tag fused in frameto its 3' end is cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag correspond to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).The infusion of HA tag to our target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding UCE 9, ATCC # 75878, is constructed by PCRusing two primers: the 5' primer 5' GATCGGATCCACCATGACAGTCCAAGCACTAG 3'(SEQ ID No. 23) contains a Bam HI site followed by 3 nucleotidesresembling a Kozak sequence plus 19 nucleotides of UCE 9 coding sequencestarting from the initiation codon; the 3' sequence 5'GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTATGTGGCGTACCGCTTGG3' (SEQ ID No.24) contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 17 nucleotides of the UCE 9 coding sequence(not including the stop codon). Therefore, the PCR product contains aBam HI site, UCE 9 coding sequence followed by HA tag fused in frame, atranslation termination stop codon next to the HA tag, and an XbaI site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith Bam HI and XbaI restriction enzyme and ligated. The ligationmixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037) the transformed culture is plated on ampicillin mediaplates and resistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant UCE 9, COS cellsare transfected with the expression vector by DEAE-DEXTRAN method (J.Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989)). The expression of the UCE9-HA protein is detected by radiolabelling and immunoprecipitationmethod (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, (1988)). Cells are labelled for 8 hours with ³⁵S-cysteine two days post transfection. Culture media are then collectedand cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40,0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al.,Id. 37:767 (1984)). Both cell lysate and culture media are precipitatedwith a HA specific monoclonal antibody. Proteins precipitated areanalyzed on 15% SDS-PAGE gels.

EXAMPLE 10 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5' and 3' end sequencesrespectively. The 5' primer containing an EcoRI site and the 3' primer$further includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified $EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 24                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 444 BASE PAIRS                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGGCTCTGAAGAGAATCCACAAGGAATTGAATGATCTGGCACGGGACCCTCCAGCACAG60                TGTTCAGCAGGTCCTGTTGGAGATGATATGTTCCATTGGCAAGCTACAATAATGGGGCCA120               AATGACAGTCCCTATCAGGGTGGAGTATTTTTCTTGACAATTCATTTCCCAACAGATTAC180               CTCTTCAAACCACCTAAGGTTGCATTTACAACAAGAATTTATCATCCAAATATTAACAGT240               AATGGCAGCATGTGTCTTGATATTCTACGATCACAGTGGTCTCCAGCACTAACTATTTCA300               AAAGTACTCTTGTCCATCTGTTCTCTGTTGTGTGATCCCAATCCAGATGATCCTTTAGTG360               CCTGAGATTGCTCGGATCTACAAAACAGATAGAGAAAAGTACAACAGAATAGCTCGGGAA420               TGGACTCAGAAGTATGCGATGTAA444                                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 147 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaLeuLysArgIleHisLysGluLeuAsnAspLeuAlaArg                                 51015                                                                         AspProProAlaGlnCysSerAlaGlyProValGlyAspAspMet                                 202530                                                                        PheHisTrpGlnAlaThrIleMetGlyProAsnAspSerProTyr                                 354045                                                                        GlnGlyGlyValPhePheLeuThrIleHisPheProThrAspTyr                                 505560                                                                        LeuPheLysProProLysValAlaPheThrThrArgIleTyrHis                                 657075                                                                        ProAsnIleAsnSerAsnGlySerMetCysLeuAspIleLeuArg                                 808590                                                                        SerGlnTrpSerProAlaLeuThrIleSerLysValIleLeuSer                                 95100105                                                                      IleCysSerLeuLeuCysAspProAsnProAspAspProLeuVal                                 110115120                                                                     ProGluIleAlaArgIleTyrLysThrAspArgGluLysTyrAsn                                 125130135                                                                     ArgIleAlaArgGluTrpThrGlnLysTyrAlaMet                                          140145                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 465 BASE PAIRS                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGGCGGCCAGCAGGAGGCTGATGAAGGAGCTTGAAGAAATCCGCAAATGTGGGATGAAA60                AACTTCCGTAACATCCAGGTTGATGAAGCTAATTTATTGACTTGGCAAGGGCTTATTGTT120               CCTGACAACCCTCCATATGATAAGGGAGCCTTCAGAATCGAAATCAACTTTCCAGCAGAG180               TACCCATTCAAACCACCGAAGATCACATTTAAAACAAAGATCTATCACCCAAACATCGAC240               GAAAAGGGGCAGGTCTGTCTGCCAGTAATTAGTGCCGAAAACTGGAAGCCAGCAACCAAA300               ACCGACCAAGTAATCCAGTCCCTCATAGCACTGGTGAATGACCCCCAGCCTGAGCACCCG360               CTTCGGGCTGACCTAGCTGAAGAATACTCTAAGGACCGTAAAAAATTCTGTAAGAATGCT420               GAAGAGTTTACAAAGAAATATGGGGAAAAGCGACCTGTGGACTAA465                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 154 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaAlaSerArgArgLeuMetLysGluLeuGluGluIleArg                                 51015                                                                         LysCysGlyHisLysAsnPheArgAsnIleGlnValAspGluAla                                 202530                                                                        AsnLeuLeuThrTrpGlnGlyLeuIleValProAspAsnProPro                                 354045                                                                        TyrAspLysGlyAlaPheArgIleGluIleAsnPheProAlaGlu                                 505560                                                                        TyrProPheLysProProLysIleThrPheLysThrLysIleTyr                                 657075                                                                        HisProAsnIleAspGluLysGlyGlnValCysLeuProValIle                                 808590                                                                        SerAlaGluAsnTrpLysProAlaThrLysThrAspGlnValIle                                 95100105                                                                      GlnSerLeuIleAlaLeuValAsnAspProGlnProGluHisPro                                 110115120                                                                     LeuArgAlaAspLeuAlaGluGluTyrSerLysAspArgLysLys                                 125130135                                                                     PheCysLysAsnAlaGluGluPheThrLysLysTyrGlyGluLys                                 140145150                                                                     ArgProValAsp                                                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 582 BASE PAIRS                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATGACAGTCCAAGCACTAGGGCACGAGAGTTCCGATGGAGATCAACGTGAAAGTGTTCAG60                CAAGAACCAGAAAGAGAACAAGTTCAGCCCAAGAAAAAGGAGGGAAAAATATCCAGCAAA120               ACCGCTGCTAAATTGTCAACTAGTGCTAAAAGAATTCAGAAGGAACTTGCAGAAATCACA180               TTGGACCCTCCTCCCAACTGTAGTGCTGGACCCAAAGGAGACAACATTTATGAATGGAGG240               TCAACTATATTGGGACCCCCAGGATCTGTCTATGAAGGAGGGGTGTTCTTTCTTGACATT300               ACCTTTTCACCAGACTATCCGTTTAAACCCCCTAAGGTTACCTTCCGAACAAGATTTTTT360               CAGTGTAATATTAACAGCCAAGGTGTGATCTGTCTGGACATCTTAAAGGACAACTGGAGT420               CCGGCTTTAACTATTTCTAAAGTTCTCCTCTCCATCTGCTCACTTCTTACAGATTGCAAC480               CCTGCTGACCCTCTGGTGGGGAGCATCGCCACACAGTACATGACCAACAGAGGAGAGCAT540               GACCGGATGGACAGACAGTGGACCAAGCGGTACGCCACATAG582                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 193 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetThrValGlnAlaLeuGlyHisGluSerSerAspGlyAspGln                                 51015                                                                         ArgGluSerValGlnGlnGluProGluArgGluGlnValGlnPro                                 202530                                                                        LysLysLysGluGlyLysIleSerSerLysThrAlaAlaLysLeu                                 354045                                                                        SerThrSerAlaLysArgIleGlnLysGluLeuAlaGluIleThr                                 505560                                                                        LeuAspProProProAsnCysSerAlaGlyProLysGlyAspAsn                                 657075                                                                        IleTyrGluTrpArgSerThrIleLeuGlyProProGlySerVal                                 808590                                                                        TyrGluGlyGlyValPhePheLeuAspIleThrPheSerProAsp                                 95100105                                                                      TyrProPheLysProProLysValThrPheArgThrArgPhePhe                                 110115120                                                                     HisCysAsnIleAsnSerGlnGlyValIleCysLeuAspIleLeu                                 125130135                                                                     LysAspTrpTrpSerProAlaLeuThrIleSerLysValLeuLeu                                 140145150                                                                     SerIleCysSerLeuLeuThrAspCysAsnProAlaAspProLeu                                 155160165                                                                     ValGlySerIleAlaThrCysTyrMetThrAsnArgGlyGluHis                                 170175180                                                                     AspArgMetAspArgGlnTrpThrLysArgTyrAlaThr                                       185190                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCCGGATCCGCTTCGAAGAGAATCCACAAG30                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCGCAAGCTTTTACATCGCATACTTCTGAGTCC33                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCCGGATCCGCGGCCAGCAGGAGGCTGATG30                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCGCAAGCTTTTAGTCCACAGGTCG25                                                   (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCGCGGATCCACAGTCCAAGCACTAGGGC29                                               (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GCGCAAGCTTCTATGTGGCGTACCGCTTGG30                                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCGCGGATCCACCATGGCTCTGAAGAGAATCC32                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GCGCTCTAGATTACATCGCATACTTCTGAGTCC33                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GCGCGGATCCACCATGGCGGCCAGCAGGAGGCT33                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GCGCTCTAGATTAGTCCACAGGTCG25                                                   (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GATCGGATCCACCATGACAGTCCAAGCACTAG32                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GCGCTCTAGACTATGTGGCGTACCGCTTGG30                                              (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GCGCGGATCCACCATGGCTCTGAAGAGAATCC32                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTACATCGCATACTTCTGAG57                   (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GCGCGGATCCACCATGGCGGCCAGCAGGAGGC32                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGTCCACAGGTCG52                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GATCGGATCCACCATGACAGTCCAAGCACTAG32                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTATGTGGCGTACCGCTTGG57                   __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide having at least a 95% identity to a member selected fromthe group consisting of:(a) a polynucleotide encoding a polypeptidecomprising amino acids 2 to 154 of SEQ ID NO:4; (b) a polynucleotideencoding a polypeptide comprising amino acids 2 to 193 of SEQ ID NO:6;and (d) the complement of (a) or (b).
 2. The isolated polynucleotide ofclaim 1 wherein said member is (a).
 3. The isolated polynucleotide ofclaim 1 wherein said member is (b).
 4. The isolated polynucleotide ofclaim 1 wherein said member is (a) and the polypeptide comprises aminoacids 1 to 154 of SEQ ID No:4.
 5. The isolated polynucleotide of claim 1wherein said member is (b) and the polypeptide comprises amino acids 1to 193 of SEQ ID No:6.
 6. The isolated polynucleotide of claim 1comprising a polynucleotide encoding a polypeptide comprising the aminoacid sequence identical to amino acids 2 to 154 of SEQ ID NO:4.
 7. Theisolated polynucleotide of claim 1 comprising a polynucleotide encodinga polypeptide comprising the amino acid sequence identical to aminoacids 2 to 193 of SEQ ID NO:6.
 8. The isolated polynucleotide of claim2, wherein the polynucleotide is DNA.
 9. The isolated polynucleotide ofclaim 3, wherein the polynucleotide is DNA.
 10. The isolatedpolynucleotide of claim 2 comprising a polynucleotide encoding apolypeptide comprising the amino sequence identical to amino acids 1 to154 of SEQ ID NO:4.
 11. The isolated polynucleotide of claim 3comprising a polynucleotide encoding a polypeptide comprising the aminosequence identical to amino acids 1 to 193 of SEQ ID NO:6.
 12. Theisolated polynucleotide of claim 2, wherein said polynucleotide is RNA.13. The isolated polynucleotide of claim 3, wherein said polynucleotideis RNA.
 14. A method of making a recombinant vector comprising insertingthe isolated polynucleotide of claim 2 into a vector, wherein saidpolynucleotide is DNA.
 15. A recombinant vector comprising thepolynucleotide of claim 2, wherein said polynucleotide is DNA.
 16. Arecombinant host cell comprising the polynucleotide of claim 2, whereinsaid polynucleotide is DNA.
 17. A method for producing a polypeptidecomprising expressing from the recombinant cell of claim 16 thepolypeptide encoded by said polynucleotide.
 18. A method of making arecombinant vector comprising inserting the isolated polynucleotide ofclaim 3 into a vector, wherein said polynucleotide is DNA.
 19. Arecombinant vector comprising the polynucleotide of claim 3, whereinsaid polynucleotide is DNA.
 20. A recombinant host cell comprising thepolynucleotide of claim 3, wherein said polynucleotide is DNA.
 21. Amethod for producing a polypeptide comprising expressing from therecombinant cell of claim 20 the polypeptide encoded by saidpolynucleotide.
 22. A process for producing a polypeptidecomprising:expressing from a recombinant cell containing thepolynucleotide of claim 6 the polypeptide encoded by saidpolynucleotide.
 23. A process for producing a polypeptidecomprising:expressing from a recombinant cell containing thepolynucleotide of claim 7 the polypeptide encoded by saidpolynucleotide.
 24. A process for producing a polypeptidecomprising:expressing from a recombinant cell containing thepolynucleotide of claim 10 the polypeptide encoded by saidpolynucleotide.
 25. A process for producing a polypeptidecomprising:expressing from a recombinant cell containing thepolynucleotide of claim 11 the polypeptide encoded by saidpolynucleotide.
 26. The isolated polynucleotide of claim 2 comprisingnucleotides 4 to 462 of SEQ ID NO:3.
 27. The isolated polynucleotide ofclaim 2 comprising nucleotides 1 to 462 of SEQ ID NO:3.
 28. The isolatedpolynucleotide of claim 2 comprising the nucleotides of the sequence ofSEQ ID NO:3.
 29. The isolated polynucleotide of claim 3 comprisingnucleotides 4 to 579 of SEQ ID NO:5.
 30. The isolated polynucleotide ofclaim 3 comprising nucleotides 1 to 579 of SEQ ID NO:5.
 31. The isolatedpolynucleotide of claim 3 comprising the nucleotides of the sequence ofSEQ ID NO:5.
 32. An isolated polynucleotide comprising a polynucleotidehaving at least a 95% identity to a member selected from the groupconsisting of:(a) a polynucleotide encoding the same mature polypeptideencoded by the human cDNA in ATCC Deposit No. 75877; and (b) thecomplement of (a).
 33. An isolated polynucleotide comprising apolynucleotide having at least a 95% identity to a member selected fromthe group consisting of:(a) a polynucleotide encoding the same maturepolypeptide encoded by the human cDNA in ATCC Deposit No. 75876; and (b)the complement of (a).
 34. The isolated polynucleotide of claim 33,wherein the member is (a).
 35. The isolated polynucleotide of claim 33,wherein said polynucleotide comprises DNA identical to the codingportion of the human cDNA in ATCC Deposit No. 75876 which encodes amature polypeptide.
 36. A process for producing a polypeptidecomprising:expressing from a recombinant cell containing the nucleotideof claim 33 the mature polypeptide encoded by said polynucleotide. 37.An isolated polynucleotide comprising a polynucleotide having at least a95% identity to a member selected from the group consisting of:(a) apolynucleotide encoding the same mature polypeptide encoded by the humancDNA in ATCC Deposit No. 75878; and (b) the complement of (a).
 38. Theisolated polynucleotide of claim 37, wherein the member is (a).
 39. Theisolated polynucleotide of claim 37, wherein said polynucleotidecomprises DNA identical to the coding portion of the human cDNA in ATCCDeposit No. 75878 which encodes a mature polypeptide.
 40. A process forproducing a polypeptide comprising:expressing from a recombinant cellcontaining the nucleotide of claim 37 the mature polypeptide encoded bysaid polynucleotide.